Our research focuses on the biochemical signaling pathways and cellular processes regulated by the Ras and Rho families of small GTPases. These proteins regulate important physiological processes in a variety of cell types, including contraction of smooth muscle cells and proliferation of epithelial cells. Abnormal signaling by these proteins may contribute to several diseases, including asthma, hypertension, atherosclerosis, and cancer. The importance of these proteins in human health provides the driving force for our studies.
Nuclear localization signals in members of the Ras and Rho families of small GTPases
A major goal of our research is to understand how small GTPases in the Ras and Rho families regulate the cytoskeletal organization, contraction, migration, and proliferation of mammalian cells. It is generally accepted that members of the Ras and Rho families regulate critical cellular functions by interacting with proteins found in the cytoplasm or at cellular membranes. However, we discovered that some members of the Rho family enter the nucleus, where these proteins may regulate nuclear functions. A specific amino acid sequence called the "nuclear localization signal" (NLS) is required for many proteins to enter the nucleus. We observed that NLS sequences are present in the C-terminal region of Rac1 and potentially other Ras and Rho family members, and are evolutionarily conserved across several phyla. We found that Rac1 uses its C-terminal NLS to undergo nucleocytoplasmic shuttling, which is a previously unsuspected function of small GTPases. The NLS may be required for the nuclear entry of small GTPases when they are associated with other proteins in complexes that are too large to passively diffuse through nuclear pores. The nuclear entry of small GTPases may allow signals that are generated in the cytoplasm to be sensed in the nucleus. This ability to enter the nucleus expands the number of signaling pathways that are potentially regulated by these small GTPases, and may explain how these proteins can participate in a wide range of both normal and abnormal cellular responses.
Regulation of small GTPases by the unique protein known as SmgGDS
The activity of small GTPases is regulated by their ability to bind GTP and hydrolyze the bound GTP to GDP. Small GTPases are more active in the GTP-bound state, and less active in the GDP-bound state. Proteins known as guanine nucleotide exchange factors (GEFs) activate a small GTPase by inducing it to bind GTP. There are over 70 known proteins that can act as GEFs for different small GTPases. We are studying one of these proteins, called SmgGDS. SmgGDS is a very unique protein because it interacts with more small GTPases than any other known GEF, but it does not possess any of the known catalytic domains found in other GEFs. Our studies indicate that SmgGDS regulates the activities of many small GTPases in the Ras and Rho families. We found that SmgGDS regulates the contraction and migration of vascular smooth muscle cells by activating the small GTPase RhoA, indicating that SmgGDS may be an important participant in diseases involving abnormal contraction and migration of vascular smooth muscle cells, such as hypertension and atherosclerosis. We also found that SmgGDS expression is elevated in several types of cancer and is needed for cancer cells to proliferate and migrate, indicating that SmgGDS is an important regulator of tumorigenesis and metastasis.
We recently discovered that there are two functionally distinct splice variants of SmgGDS, which we named SmgGDS-607 and SmgGDS-558, based on the number of amino acids in each protein. We found that these SmgGDS splice variants participate in signaling pathways that regulate the prenylation of small GTPases. Prenylation is the covalent attachment of an isoprenyl group (farnesyl or geranylgeranyl) to the C-terminus of the small GTPase. This post-translational modification increases the association of small GTPases with cell membranes, where small GTPases are believed to be most active. We found that in mammalian cells, SmgGDS-607 regulates the entry of non-prenylated small GTPases into the prenylation pathway, whereas SmgGDS-558 promotes the trafficking of prenylated small GTPases to cell membranes. These different functions of SmgGDS splice variants provide a previously unsuspected mechanism to control small GTPase prenylation and localization. The participation of SmgGDS splice variants in the prenylation pathway explains how SmgGDS can regulate the activities of so many different small GTPases. We are currently examining how SmgGDS splice variants interact with different small GTPases to regulate their prenylation and trafficking. These studies will further define the unique roles of SmgGDS in cardiovascular disease and cancer.
Berg, T. J., Gastonguay, A. J., Lorimer, E. L., Kuhnmuench, J. R., Li, R., Fields, A. P., and C. L. Williams. 2010. Splice variants of SmgGDS control small GTPase prenylation and membrane localization. J. Biol. Chem. 285:35255-35266.
Zhi, H., Yang, X. J., Kuhnmuench, J., Berg, T., Thill, R., Yang, H., See, W. A., Becker, C. G., Williams, C. L., and R. Li. 2009. SmgGDS is upregulated in prostate carcinoma and promotes tumor phenotypes in prostate cancer cells. J. Pathol. 217:389-397.
Thill, R., Campbell, W., and C. L. Williams. 2008. Identification and characterization of the unique guanine nucleotide exchange factor, SmgGDS, in vascular smooth muscle cells. J. Cell. Biochem. 104:1760-1770.
Tew, G. W., Lorimer, E. S., Zhi, H., Li, R., and C. L. Williams. 2008. SmgGDS regulates cell proliferation, migration, and NF-κB transcriptional activity in non-small cell lung carcinoma. J. Biol. Chem. 283:963-976.
Endsley, M., Thill, R., Choudhry, I., Williams, C. L., Kajdacsy-Balla, A., Campbell, W. C., and K. Nithipatikom. 2008. Expression and function of fatty acid amide hydrolase in prostate cancer Int. J. Cancer 123:1318-1326.
Kotamraju, S., Williams, C. L., and B. Kalyanaraman. 2007. Statin-induced breast cancer cell death: role of inducible nitric oxide and arginase-dependent pathways. Cancer Res. 67:7386-7394.
Zhang Y., Deng, Q., Porath, J. A., Williams, C. L., Pederson-Gulrud, K. J., and J. T. Barbieri. 2007. Plasma membrane localization affects the RhoGAP specificity of Pseudomonas ExoS. Cell Microbiol. 9:2192-2201.
Lanning, C. C., Daddona, J. L., Ruiz-Velasco, R., Shafer, S. H., and C. L. Williams. 2004. The Rac1 C-terminal polybasic region regulates the nuclear localization and protein degradation of Rac1. J. Biol. Chem. 279:44197-44210, 2004.
Shafer, S. H. and C. L. Williams. 2004. Elevated Rac1 activity changes the M3 muscarinic acetylcholine receptor-mediated inhibition of proliferation to induction of cell death. Mol. Pharmacol. 65:1080-1091.
Williams, C. L. 2003. The polybasic region of Ras and Rho family members: A regulator of protein interactions and membrane association and a site of nuclear localization signal sequences. Cellular Signaling 15:1071-1080.
Lanning, C. C., Ruiz-Velasco, R., and C. L. Williams. 2003. Novel mechanism of the co-regulation of nuclear transport of SmgGDS and Rac1. Biol. Chem. 278:12495-12506.
Varker, K., S. Phelps, M. King, and C. L. Williams. 2003. The small GTPase RhoA has greater expression in small cell lung carcinoma than in non-small cell lung carcinoma and contributes to their unique morphologies. Int. J. Oncol. 22:671-681.
Shafer, S. H. and C. L. Williams. 2003. Non-small and small cell lung carcinoma cell lines exhibit cell type-specific sensitivity to edelfosine-induced cell death and different cell line-specific responses to edelfosine treatment. Int. J. Oncol. 23:389-400.